Differential Effects of K<sup>+</sup> Channel Blockers on Phasic Contractility of Transverse and Longitudinal Rat Detrusor Strips

doi:10.4236/oju.2011.13013

Paper Menu >>

Journal Menu >>

Open Journal of Urology, 2011, 1, 60-65

doi:10.4236/oju.2011.13013 Published Online August 2011 (http://www.SciRP.org/journal/oju)

Differential Effects of K+ Channel Blockers on

Phasic Contractility of Transverse and Longitudinal

Rat Detrusor Strips

Aneira Gracia Hidayat Santoso1, Wan Ning Lo1, Wil lma nn Liang1,2

1School of Biological Sciences, College of Science, Nanyang

Technologi cal University , Singapore City, Singapor e

2Institute of Advanced Studies, Nanyang Technolo gi cal

University, Singapore City, Singapore

E-mail: [email protected]

Received May 30, 201 1; revised June 28, 2011; accepted July 5, 2011

Abstract

Spontaneous phasic contractions of detrusor smooth muscle are pivotal to the normal bladder filling process.

The role of K+ channels in mediating phasic contractions has been investigated on different occasions, but

only in detrusor strips isolated longitudinally. In this study, the effects of individual K+ blockers were exam-

ined in both transverse and longitudinal detrusor strips. Detrusor strips were isolated transversely and longi-

tudinally from young adult rat bladders. Tension before and after the introduction of K+ channel blockers

was measured using a myograph. Phasic activity was determined by calculating the integral of tension fluc-

tuations. Phasic activity of transverse strips was increased under tetraethylammonium chloride (TEA),

4-aminopyridine (4-AP) and iberiotoxin (IbTx) treatments. Longitudinal phasic activity was increased under

charybdotoxin (ChTx) treatment. Neither glibenclamide (Glib) nor apamin treatment elicited any significant

effect in both transverse and longitudinal phasic activity. The results indicated that phasic activity was medi-

ated differently depending on the contractile direction. Data from this study reiterate that in addition to the

conventional longitudinal direction, the transverse direction also presents significance when examining the

contractility of a sac-like organ like the bladder.

Keywords: Detrusor Smooth Muscle, Contractility, K+ Channel, Transverse, Longitudinal

1. Introduction

Urine release and storage are major functions of the uri-

nary bladder. Forceful contractions of the detrusor

smooth muscle are essential for urine release. During

bladder filling, detrusor wall tension and intravesical

pressure fluctuate to adjust to the changing urine volume

[1]. The ever changing contractility of the detrusor

smooth muscle contributes to spontaneous phasic con-

tractions frequently seen in whole bladder or isolated

strip experiments. Regulation of phasic contractions can

be of several origins, from nerves, the urothelium or

within the smooth muscle [2]. The importance of phasic

activity in the bladder is implicated in various disease

conditions [2-4]. Indeed many groups have investigated

one aspect or another of phasic contractions, both in

normal and diseased bladders. One group of molecular

candidates believed to mediate phasic contractions is the

K+ channels. Several groups have studied the effects of

K+ channel blockers and reached variable findings attrib-

utable to differences between species and experimental

conditions [5-9]. Although not as extensively used as

other animals, the rat remains a valuable model for stud-

ying bladder physiology. Diseased bladder models have

been established in the rat [4,10]. Findings from the

normal rat bladder therefore provide good references to

identify diseased-induced changes in disease models.

The vast majority of literature on bladder contractility of

the rat (or other animals) reported on longitudinal con-

tractions only, irrespective of contractile differences be-

tween transverse and longitudinal directions [11-15].

These differences point to the importance in considering

contractions in more than one direction. Thus, this study

is conducted to identify differential modulation by K+

A. G. H. SANTOSO ET AL.

channels in detrusor phasic contractions between trans-

verse and longitudinal directions.

2. Materials and Methods

2.1. Tissue Preparation

All procedures were performed according to rules out-

lined by the Institutional Animal Care and Use Commit-

tee at Nanyang Technological University, Singapore

(Project approval No.: ARF SBS/NIE-A 003). Six- to

seven-week-old Sprague-Dawley rats of either gender

were killed by CO2 asphyxiation. Forty-nine rats were

used in total, from which 40 transverse and 30 longitu-

dinal strips were isolated (see below). The whole bladder

was harvested as previously described and immediately

placed in carbogen-aerated ice-cold Krebs’ solution [11].

The bladder base, which made up about one third of the

bladder, was discarded. Only tissues isolated from the

bladder dome (detrusor) were used. The bladder dome

was cut open along the lateral sides and the urothelium

was exposed. Fine pins were used to fix the tissue on a

Sylgard®-coated petri dish. Using a razor blade, two

strips measuring 5 mm by 1 mm each were dissected

from the detrusor. One strip was cut with the longer side

parallel to the longitud inal axis of the detrusor. Th e other

strip was cut with the longer side parallel to the trans-

verse axis of the detrusor. The longer side of the strip

was in line with the direction of contractile force meas-

urements as previously done [12]. The urothelium was

kept intact. All strips were mounted on a tissue myog-

raph system (Danish Myo Technology Model 800MS,

Aarhus, Denmark) containing Krebs’ solution at 37˚C.

Isometric tension was monitored in both transverse and

longitudinal directions and recorded using a Powerlab

interface and the LabChart software (ADInstruments,

Bella Vista, Australia).

2.2. Experimental Protocol

The detrusor strips were allowed to equilibrate for 30

minutes with multiple washouts at 2 g of resting tension.

The viability of the strips was tested using K+-Krebs’

solution bubbled with a mixture of 95% oxygen and 5%

carbon dioxide. After another 30 minutes of continuous

washout, a K+ channel blocker was added. The effective

concentration of each K+ channel blocker in rat detrusor

strips was pre-determined in previous studies [12,13].

Blockers used were as follows (concentrations in paren-

theses): tetraethylammonium chloride (TEA, 10 mM),

4-aminopyridine (4-AP, 3 mM), glibenclamide (Glib,

0.1 µM), iberiotoxin (IbTx, 0.1 µM), charybdotoxin

(ChTx, 0.1 µM) and apamin (0.1 µM). The incubation

period of each K+ channel blocker was 10 minutes.

2.3. Drugs and Chemicals

The composition of Krebs’ solution was as follows (in

mM): NaCl (119), MgCl2 (1.2), NaH 2PO4 (1.2), NaH CO3

(15), KCl (4 .6), CaCl2 (1.5), Glucose (11). For K+-Krebs’

solution, no NaCl was added but 124 mM KCl was used

instead. All constituents remained the same otherwise.

All chemicals and drugs used in this study were pur-

chased from Sigma-Aldrich Co. (Singapore, Singapore).

All drugs were dissolved in Ca2+-free Krebs’ solution

except 4-AP (in 70% ethanol), Glib (in dimethyl sul-

fonide) and apamin (in 0.05 M acetic acid). For drugs not

dissolved in Ca2+-free Krebs’ solution, a maximum of

1-to-1000 dilution from the stock drug solution was per-

formed to prevent nonspecific tissue effects due to the

solvents.

2.4. Data and Statistical Analysis

Sample tracings were shown in pairs, consisting of con-

trol and K+ blocker-treated readings from the same de-

trusor strip. Fluctuations in tension (F) were expressed

as a percentage of the maximal contraction (F) to

K+-Krebs’ solution. According to the literature, the inte-

gral under the tension recording curve could give an in-

dication of the amount of phasic activity [11,16]. In this

study, phasic activity was quantified this way for a

2-minute period before (i.e. control) and after the addi-

tion of each K+ blocker. For K+ channel blocker-medi-

ated phasic activity, readings were taken starting at 10

minutes after the addition of the blocker. Phasic activity

in the presence of the K+ channel blocker was expressed

as a percent change from the control phasic activity. Sta-

tistical analysis was done using the Prism 4 software

(GraphPad Software Inc., La Jolla, CA, USA). Student’s

t-test was used to determine if the percent change in pha-

sic activity was significantly deviated from zero, which

was the control level by definition. The difference be-

tween the percent change in transverse and longitudinal

phasic activity was also determined by Student’s t-test.

All data shown in graph s were mean ± SEM. P values of

less than 0.05 (P < 0.05) were considered to be statisti-

cally different.

3. Results

3.1. Transverse Phasic Activity Was More

Sensitive to Blockade of Voltage-Sensitive

K+ Channels

Tetraethylammonium chloride (TEA) is a nonselective

62 A. G. H. SANTOSO ET AL.

blocker of voltage-sensitive K+ (Kv), ATP-sensitive K+

(KATP) and large-conductance Ca2+-activated K+ (BK)

channels. Figure 1(a) shows sample tracings of phasic

activity before (i.e. control) and after adding the K+

channel blockers. In both transverse and longitudinal

directions, phasic activity was significantly increased by

10 mM TEA treatment (Figure 1(b)). Transverse phasic

activity was increased by 110% ± 20%, compared to a

60% ± 10% increase in longitudinal phasic activity (P <

0.05). Since TEA could act at more than one type of K+

channels, namely Kv, KATP and BK channels, selective

blockade of Kv and KATP channels, by 4-aminopyridine

(4-AP) and glibenclamide (Glib) respectively, was ex-

amined next. In the presence of 3 mM 4-AP, transverse

phasic activity was significantly increased (by 50% ±

20%) whereas that of longitudinal remained unchanged

from control (10% ± 10%) (Figure 1(b)). Similar to TEA

treatment, 4-AP elicited a greater effect in transverse

than longitudinal phasic activity (P < 0.05). Under 0.1 M

Glib treatment, there was a small but not significant de-

crease in both transverse and longitudinal phasic activity,

respectively, by 30% ± 10% and 20% ± 20% (Figure

1(b)). The function of KATP channels in mediating phasic

activity was not implicated, whereas that of Kv channels

was demonstrated in the transverse contractile direction.

3.2. Blockade of Ca2+-Activated K+ Channels

Revealed Differential Effects in Transverse

and Longitudinal Phasic Activity

Figure 2(a) shows sample tracings of phasic activity

before and after adding the various Ca2+-activated K+

channel blockers. Aside from Kv and KATP channels, BK

channels are also blocked by TEA. Iberiotoxin (IbTx)

was used here to block BK channel activity selectively.

Only transverse phasic activity was significantly in-

creased (by 80% ± 20% from the control level) under 0.1

M IbTx treatment (Figure 2(b)). As with TEA and

4-AP, IbTx also elicited a greater stimulatory effect in

transverse than longitudinal phasic activity (P < 0.05).

Charybdotoxin (ChTx) is a blocker of intermediate-

conductance Ca2+-activated K+ (IK) channels. In the

presence of 0.1 M ChTx, longitudinal phasic activity

was significantly increased from the control level by

60% ± 20% (Figure 2(b)). The greater effect elicited by

ChTx in longitudinal phasic activity was approaching

statistical significance (P = 0.0765). After adding 0.1 M

apamin, a selective blocker of small-conductance Ca2+-

activated K+ (SK) channels, both transverse and longitu-

dinal phasic activity was only modestly suppressed, by

20% ± 10% and 20% ± 9% (Figure 2(b)). The findings

here suggested that both BK and IK channels mediated

phasic activity, but did so distinctly according to the con-

(a)

(b)

Figure 1. Transverse and longitudinal phasic contractions

under the influence of nonselective and selective voltage-

sensitive K+ (Kv) and ATP-sensitive K+ (KATP) channel

blockers. (a) Representative tracings of transverse (Tr) and

longitudinal (Lg) phasic contractile activity before (i.e. con-

trol) and after treatment with 10 mM tetraethylammonium

chloride (TEA) (Tr: n = 4; Lg: n = 4), 3 mM 4-amino- pyri-

dine (4-AP) (Tr: n = 8; Lg: n = 5) or 0.1 M glibenclamide

(Glib) (Tr: n = 6; Lg: n = 5); (b) Percent change in trans-

verse (solid columns) and longitudinal (open columns) pha-

sic activity from the control level. Significantly higher pha-

sic activity was present in the transverse direction under

TEA and 4-AP treatments. *denotes P < 0.05 vs. control

level at 0%. #denotes P < 0.05 between transverse and lon-

gitudinal.

tractile direction.

4. Discussion

The role of K+ channels in mediatin g spontaneous ph asic

contractions in the detrusor have been studied by differ-

ent groups over the recent years. Both urothelium-intact

and -denuded detrusor tissues from the bladder of dif-

ferent species have been used, often yielding conflicting

results [5,6,8,12,13]. Thus, generalizations, if any, about

K+-channel-mediated phasic contractions in the detrusor

are yet to be made. Using selective K+ channel blockers

at effective concentrations previously established, we

hereby examined phasic contractile activity in both

transverse and longitudinal directions using normal adult

rat urothelium-intact detrusor strips. Despite anatomical

differences between human and rat detrusor, the latter is

still of value due to the numerous established bladder

diseased models in the rat. Examples include the overact-

A. G. H. SANTOSO ET AL.

(a)

(b)

Figure 2. Transverse and longitudinal phasic contractions

under the influence of selective Ca2+-activated K+ channel

blockers. (a) Representative tracings of transverse (Tr) and

longitudinal (Lg) phasic contractile activity before (i.e. con-

trol) and after treatment with 0.1 M iberiotoxin (IbTx) (Tr:

n = 8; Lg: n = 6), 0.1 M charybdotoxin (ChTx) (Tr: n = 8;

Lg: n = 5) or 0.1 M apamin (Tr: n = 6; Lg: n = 5); (b) Per-

cent change in transverse (solid columns) and longitudinal

(open columns) phasic activity from the control level. Tran-

sverse phasic activity was increased under IbTx treatment

whereas that of longitudinal was increased in the presence

of ChTx. *denotes P < 0.05 vs. control level at 0%. #denotes

P < 0.05 between transverse and longitudinal.

tive bladder model in spontaneously hypertensive rats

and the rat bladder outlet obstruction model [4,10].

Findings in this study would lay the groundwork for

comparisons with the contractility of diseased rat detru-

sor, which would in turn give insights into altered physi-

ology in the human bladder in the future.

Whereas most other studies measured detrusor strip

contractility in the longitudinal direction, we also con-

sidered transverse contractions in our experiments. Dif-

ferences in contractile directions, i.e. transverse vs. lon-

gitudinal, in the detrusor have been reported under dif-

ferent experimental conditions [11-15]. Choice of species

and method of tissue preparation (i.e. urothelium-intact

vs. denuded tissues, or isolated strips vs. whole bladders)

apparently resulted in contrasting findings by different

groups. Nevertheless we demonstrated in the present

study that selected K+ channel blocker treatments could

reveal directional phasic contractile differences. These

differences should be considered when attempting to

draw conclusions from contractile data of a single direc-

tion only.

The stimulatory effect of various K+ channel blockers

in phasic contractions has been well documented, indi-

cating the role of K+ channels in mediating the tension

fluctuations [1]. In the basal or unstimulated state, KATP

and SK channels had no significant functional role in

longitudinal phasic activity of urothelium-intact detrusor

[5,6], findings supported by our data as well. We further

showed that transverse phasic activity was equally unaf-

fected by KATP and SK channel blockade. The distinctive

effects in transverse and longitud inal phasic activity were

demonstrated by blocking Kv, BK and IK channels. The

nonselective K+ channel blocker TEA enhanced phasic

activity in both transverse and longitudinal directions.

The effect in transverse phasic activity was greater due to

possibly larger contribution by Kv and BK channels, both

of which blocked by TEA, in this direction. This was

supported by the results of 4-AP and IbTx treatments

where transverse phasic activity was significantly higher

than the control level. The more prevalent transverse

phasic activity seen under 4-AP treatment has been

demonstrated elsewhere [12]. For IbTx, others have

shown different results depending on tissue origins and

preparation. In the longitudinal guinea-pig detrusor, pha-

sic activity was increased under IbTx treatment [8]. This

was in contrast to the whole rat detrusor where no cha-

nge in phasic activity was detected [9]. It is possible that

phasic activity in individual smooth muscle bundles may

not influence intravesical pressure in the whole bladder

to a great extent. Nevertheless, an intrinsic function in

the greater sensitivity of transverse phasic activity to-

ward BK, and also Kv channel blockade may be impli-

cated. Although ChTx could block BK channels, its use

as an IK channel blocker has been documented. In the

longitudinal but not transverse direction, phasic activity

was enhanced under ChTx treatment, again suggesting

direction-dependent differences in the regulation of pha-

sic contractions.

Spontaneous detrusor phasic contractions occur during

bladder filling to allow maintenance of bladder shape

and wall tension without drastically increasing intravesi-

cal pressure [1]. This prevents immature voiding as well

as ensures efficient micturition when necessary. Other

than K+ channels as demonstrated in this and other stud-

ies, Ca2+ channels [3], gap junctions [3] and the urothe-

lium [5,17,18] also play a part in mediating phasic con-

tractions. The role of the mucosal layer (including the

urothelium and myofibroblasts) is of particular interest

especially in disease conditions [2]. The use of normal

rat detrusor here nevertheless serves as a starting point to

examine disease-induced changes in bladders from es-

tablished rat models. Regardless of species and experi-

mental variations, the urothelium is believed to be an

active participant of normal and diseased bladder physi-

ology. Studies comparing the role of K+ channels in me-

64 A. G. H. SANTOSO ET AL.

diating urothelium-dependent and urothelium-indepen-

dent detrusor phasic contractions may be useful. It is not

possible to isolate the effects of the urothelium from

those of the smooth muscle if the intravesical pressure of

the whole bladder is measured, although this method

resembles in vivo physi ol o gy more closely.

In summary, the role of individual K+ channel block-

ers in mediating phasic activity in the detrusor was ex-

amined. In measuring phasic contractions in two direc-

tions, transversely and longidutinally, differential sensi-

tivity to the K+ channel blockers was demonstrated. Pha-

sic activity in the transverse direction could be distin-

guished from that in the longitudinal direction by using

selective blockers of Kv, BK and IK channels. The dis-

covery of differential phasic activity highlights the po-

tential importance in considering the physiological func-

tion of contractility in more than one direction.

5. Acknowledgements

This study was supported by the Singapore Ministry of

Education (RG63/06 and RG83/07) and the Institute of

Advanced Studies, Nanyang Technological University,

Singapore.

6. References

[1] A. F. Brading, “Spontaneous Activity of Lower Urinary

Tract Smooth Muscles: Correlation between Ion Chan-

nels and Tissue Function,” Journal of Physiology, Vol.

570, No. 1, 2006, pp. 13-22.

doi:10.1113/jphysiol.2005.097311

[2] C. H. Fry, E. Meng and J. S. Young, “The Physiological

Function of Lower Urinary Tract Smooth Muscle,” Au-

tonomic Neuroscience, Vol. 154, No. 1-2, 2010, pp. 3-13.

doi:10.1016/j.autneu.2009.10.006

[3] C. Fry, G.-P. Sui, N. J. Severs and C. Wu, “Spontaneous

Activity and Electrical Coupling in Human Detrusor

Smooth Muscle: Implications for Detrusor Overactivity?”

Urology, Vol. 63, No. 3, 2004, pp. 3-10.

doi:10.1016/j.urology.2003.11.005

[4] M. C. Michel and M. M. Barendrecht, “Physiological and

Pathological Regulation of the Autonomic Control of

Urinary Bladder Contractility,” Pharmacology and The-

rapeutics, Vol. 117, No. 3, 2008, pp. 297-312.

doi:10.1016/j.pharmthera.2007.12.001

[5] H. Akino, C. R. Chapple, N. McKay, R. L. Cross, S. Mu-

rakami, O. Yokoyama, R. Chess-Williams and D. J. Sell-

ers, “Spontaneous Contractions of the Pig Urinary Blad-

der: The Effect of ATP-Sensitive Potassium Channels

and the Role of the Mucosa,” British Journal of Urology

International, Vol. 102, No. 9, 2008, pp. 1168-1174.

doi:10.1111/j.1464-410X.2008.07782.x

[6] M. Ekman, K.-E. Andersson and A. Arner, “Signal

Transduction Pathways of Muscarinic Receptor Mediated

Activation in the Newborn and Adult Mouse Urinary

Bladder,” British Journal of Urology International, Vol.

103, No. 1, 2008, pp. 90-97.

doi:10.1111/j.1464-410X.2008.07935.x

[7] G. M. Herrera, T. J. Heppner and M. T. Nelson, “Regula-

tion of Urinary Bladder Smooth Muscle Contractions by

Ryanodine Receptors and BK and SK Channels,” Ameri-

can Journal of Physiology, Vol. 279, No. 1, 2000, pp.

R60-R68.

[8] T. Imai, T. Okamoto, Y. Yamamoto, H. Tanaka, K. Ko-

ike, K. Shigenobu and Y. Tanaka, “Effects of Different

Types of K+ Channel Modulators on the Spontaneous

Myogenic Contraction of Guinea-Pig Urinary Bladder

Smooth Muscle,” Acta Physiologica, Vol. 173, No. 3,

2001, pp. 323-333.

[9] Y.-K. Ng, W. C. de Groat and H.-Y. Wu, “Smooth Mus-

cle and Neural Mechanisms Contributing to the Down-

regulation of Neonatal Rat Spontaneous Bladder Contrac-

tions during Postnatal Development,” American Journal

of Physiology, Vol. 292, No. 5, 2007, pp. R2100-R2112.

[10] G. McMurray, J. H. Casey and A. M. Naylor, “Animal

Models in Urological Disease and Sexual Dysfunction,”

British Journal of Pharmacology, Vol. 147, Suppl. 2, 2006,

pp. S62-S79. doi:10.1038/sj.bjp.0706630

[11] W. Liang and W. N. Lo, “



-Adrenoceptor-Mediated Dif-

ferences in Transverse and Longitudinal Strips from the

Rat Detrusor,” International Urology and Nephrology,

Vol. 43, No. 1, 2010, pp. 99-107.

doi:10.1007/s11255-010-9759-y

[12] W. N. Lo and W. Liang, “Blockade of Voltage-Sensitive

K+ Channels Increases Contractility More in Transverse

than in Longitudinal Rat Detrusor Strips,” Urology, Vol.

73, No. 2, 2009, pp. 400-404.

doi:10.1016/j.urology.2008.08.465

[13] W. N. Lo, A. G. H. Santoso and W. Liang, “Differences

in Transverse and Longitudinal Rat Detrusor Contractility

under K+ Channel Blockade,” UroToday International

Journal, Vol. 3, No. 2, 2010, on Line.

[14] M. Pagala, D. S. Lehman, M. P. Morgan, J. Jedwab and

G. J. Wise, “Physiological Fatigue of Smooth Muscle

Contractions in Rat Urinary Bladder,” British Journal of

Urology International, Vol. 97, No. 5, 2006, pp. 1087-

1093. doi:10.1111/j.1464-410X.2006.06136.x

[15] M. K. Pagala, L. Tetsoti, D. Nagpal and G. J. Wise, “Ag-

ing Effects on Contractility of Longitudinal and Circular

Detrusor and Trigone of Rat Bladder,” Journal of Urol-

ogy, Vol. 166, No. 2, 2001, pp. 721-727.

doi:10.1016/S0022-5347(05)66050-8

[16] A. Deba, S. Palea, C. Rouget, T. D. Westfall and P. Lluel,

“Involvement of



3 Adrenoceptors in Mouse Urinary

Bladder Function: Role in Detrusor Muscle Relaxation

and Micturition Reflex,” European Journal of Pharma-

cology, Vol. 618, No. 1-3, 2009, pp. 76-83.

doi:10.1016/j.ejphar.2009.07.012

[17] K. M. Azadzoi, V. K. Heim, T. Tarcan and M. B. Siroky,

“Alteration of Urothelial-Mediated Tone in the Ischemic

Bladder: Role of Eicosanoids,” Neurourology and Uro-

dynamics, Vol. 23, No. 3, 2004, pp. 258-264.

doi:10.1002/nau.20029

A. G. H. SANTOSO ET AL.

[18] J. I. Gillespie and M. J. Drake, “The Actions of Sodium

Nitroprusside and the Phosphodiesterase Inhibitor Dipy-

ridamole on Phasic Activity in the Isolated Guinea-Pig

Bladder,” British Journal of Urology International, Vol.

93, No. 6, 2004, pp. 851-858.

doi:10.1111/j.1464-410X.2003.04727.x